The clinical implementation of molecularly targeted imaging modalities has progressed considerably in recent years. Fluorophores in the near-IR range are of particular interest for in vivo optical imaging due to the significant tissue penetration of light in this range. Despite a central role in modern biology and medicine, the compounds employed in near-IR fluorescence techniques have changed little in recent decades. Using molecular design concepts borrowed from related fields (e.g. medicinal chemistry and modern organic synthesis), we seek to develop new agents with improved utility for cancer-related imaging and microscopy. The long-term goal is to identify readily synthesized, stable, and bright fluorophores with optimal properties for biomedical imaging. Our current efforts in this area are split in two aims. Aim 1 - Synthetic methods to prepare heptamethine cyanine fluorophores. The heptamethine cyanine class of near-IR fluorophores are used for many applications, with extensive recent progress in the context of fluorescence-guided surgery. We have developed a new rearrangement reaction that enables the synthesis of previously inaccessible variants. Compared to existing agents, the compounds we have prepared exhibit improved optical properties and significantly greater chemical stability to biological nucleophiles. We have shown that these new agents can be conjugated to the antibodies and resulting conjugates display exhibit excellent fluorescence properties in microscopy experiments and in vivo animal studies. We have also shown that small changes in the polar functional groups appended to these fluorophores can have a dramatic impact on biodistrubution and tumor accumulation. We are currently pursuing approaches to improve other aspects of the optical properties of these molecules, especially their brightness and absorption profile. Aim 2 - Synthesis and evaluation of novel far-red probes. We have developed a chemical strategy to assemble polycyclic pentamethine cyanines through a cross metathesis/polycylcization strategy. When compared to conventional pentamethine cyanines, the resulting compounds exhibit significantly higher fluorescence quantum yield (4X) and, additionally, recover from sodium borohydride reduction with improved efficiency. These features allow these compounds to be used for super resolution microscopy and enable excellent photon counts without recourse to complex deoxygenation buffers. In ongoing efforts, we are developing targetable small molecules with enhanced solubility.